Dielectrophoretic cell capture

ABSTRACT

Various aspects are described for selectivity capturing cells or bioparticles on designated surfaces in dielectrophoretic systems and processes. A particular adhesive composition is described for enhancing cell retention. In addition, certain permeable polyester membranes used in the systems and processes are also described.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional application Ser.No. 61/537,179 filed on Sep. 21, 2011.

FIELD

The present subject matter relates to components, compositions, systems,and methods for selectively capturing and retaining cells andbioparticles using dielectrophoresis.

BACKGROUND

The integration of tools for manipulating and controlling cells withinmicrofluidic systems has steadily grown due to various unique featuresthat microscale technologies can provide in terms of fine control overcellular microenvironments, flow conditions, and precise cellpositioning for specific cell-cell interactions. The combination of suchtools with microsystems has enabled the study of cellular processes thatotherwise would not have been possible. Among the tools currentlyavailable to position cells in precise locations on a substrate isdielectrophoresis (DEP), which is an electrokinetic technique that cantrap particles (e.g. cells) based on polarizability differences betweenthe particle and the media in which the particles are suspended whenboth are exposed to a non-uniform field. The use of DEP has beenprimarily limited to, short-term manipulation studies of cells orpreparative methods to separate cells from complex mixtures. Few studieshave demonstrated DEP trapping for long term cell experiments where cellfunction still remains days after the trapping is effected. Therefore,it is of paramount importance, when developing DEP devices for in vitrocell studies, to demonstrate that cell viability and cell function(e.g., proliferation, motility, differentiation) are maintained afterthe electrokinetic manipulation.

A typical design for using DEP to trap cells is the placement of DEPelectrodes under a fluid flow in a microfluidic device. This arrangementallows for increased trapping of cells in a short time and the removalof untrapped cells from non-DEP parts of a substrate surface. Achallenge to this design is retaining the trapped cells in a fluid flowfield at the selected positions when the DEP forces are removed. Inorder to produce DEP forces capable of moving cells up the fieldgradient, known as positive DEP (pDEP), cells must be suspended insucrose or other low conductivity media. As opposed to cells suspendedin high conductivity media (e.g. cell growth media), pDEP conditionsproduce stronger traps, thus attracting more cells and holding them onthe substrate while the DEP forces remain active. The difficulty withthis arrangement occurs when the DEP forces are switched off and thefluid flow field dislodges the positioned cells. In order to maintainthe cells in position one needs to have good control over flow so thatcells may attach through their integrins or other adhesive proteins overa period of time. An alternative to controlling the flow by pumps andvalve systems is to have a “sticky” surface to which cells will anchorto, immediately after DEP trapping is achieved. By taking advantage ofthe extracellular molecules around the cells, such as antibodies orglycoproteins, either specific or non-specific binding can be effected.In turn, this can produce cell attachment on the pretreated surface viaantibody/antigen binding or electrostatic interactions. The latterapproach has been investigated using polyelectrolyte multiple layers(PEMs) as the surface coating material and has been shown to work whenanchoring cells for short term experiments. However, a more relevantmaterial is needed for in vitro long term cell experiments not only tofacilitate cell anchoring, but also to maintain cell proliferation andcell function.

The present inventors have demonstrated cell patterning using PEMs whenseeded in cell culture medium as well as when trapped under DEPconditions. Cells trapped under DEP and PEM conditions showed that over93% of the cells remained anchored on the PEMs after the electrodes werede-energized. However, further research has been conducted in an attemptto extend the use of this approach for long term cell experiments. Theresults obtained using PEMs and DEP conditions show deleterious effectson the cells 24 hours after DEP cell trapping (see FIGS. 1A and 1B).Specifically, referring to FIGS. 1A and 1B, P19 cells attached onto PEMsare shown under different conditions. FIG. 1A illustrates P19 cells 24hours after DEP trapping in a microfluidic channel. Cells were anchoredon PEMs while in sucrose media, and then the sucrose was replaced withcell growth media. The faint vertical lines in the center are the indiumtin oxide (ITO) electrodes used for DEP trapping. FIG. 1B illustratesP19 cells 24 hours after seeded on PEMs in cell growth media. Note thedifference between P19 cells poorly attached in FIG. 1A versuswell-attached healthy cells shown in FIG. 1B. The scale bar in FIG. 1Ais 100 μm. Therefore, an alternative to this approach is needed toachieve long term cell experiments using a “sticky” surface and DEP.

The precise positioning of cells is a key requirement when utilizingmicrofluidic systems, specifically when cells are needed to be indefined areas for their stimulation and study. A number of approacheshave been introduced to manipulate or capture cells withinmicrochannels. These aproaches vary from mechanical traps and flowcontrol, to optical and electronic techniques. Among the electronictechniques, dielectrophoresis (DEP) has gained much attention in themicrofluidics community. This phenomenon was first described by Pohl in1951. However, it was not until the last decade that the number ofpublications increased significantly for applications includingbiosensors, medical diagnostics, particle filtration, nanoassembly, andDNA manipulation. The main advantages that DEP offers for particlemanipulation include label free entrapment, simplicity ofinstrumentation, favorable scaling effects, the ability to applyrepulsive (negative DEP) and attractive (positive DEP) forces, and thelack of microfabricated obstacles that distort the flow within thechannels. DEP coupled with lab-on-a-chip devices have demonstratedsuitability for DEP-based cell applications such as separation by size,sorting, focusing, filtration, trapping, and patterning.

In general, DEP electrodes have been patterned on solid substrates suchas glass slides and silicon wafers. However, few previous efforts haveinvestigated patterning electrodes, for purposes other than DEP, ontopermeable surfaces. For example, Duan and Meyerhoff showed thatmetallization of permeable membranes was possible and used patternednylon membranes for sandwich enzyme immunoassays. Later, {hacek over(S)}vor{hacek over (c)}ik et al. characterized the sputtering process tometallize polyethylene terephthalate (PET).

SUMMARY

The difficulties and drawbacks associated with previously knowntechnology are addressed in the present compositions, assemblies andmethods for capturing bioparticles.

In one aspect, the present subject matter provides a layered compositionfor capturing bioparticles during dielectrophoresis (DEP). The layeredcomposition comprises at least one layer of an adhesion material (i.e.extracellular matrix), and a layer of a polycation material disposed onthe at least one layer of the adhesion material. The layer of thepolycation material provides an exposed face for capturing bioparticlesduring dielectrophoresis.

In another aspect, the present subject matter provides a layeredassembly for use in dielectrophoresis (DEP). The assembly comprises apolyester permeable membrane defining an outer face, at least oneelectrically conductive member disposed on the outer face of themembrane, and a layered composition disposed on at least one of theouter face of the membrane and the at least one electrically conductivemember. The layered composition includes (i) at least one layer of anadhesion material, and (ii) a layer of a polycation material disposed onthe at least one layer of the adhesion material.

In still another aspect, the present subject matter provides a methodfor retaining bioparticles along a target surface duringdielectrophoresis (DEP). The method comprises providing a system forperforming dielectrophoresis including provisions for generating anon-uniform electric field proximate the target surface. The method alsocomprises applying a layered composition on the target surface. Thelayered composition includes (i) at least one layer of an adhesionmaterial and (ii) a layer of a polycation material disposed on the atleast one layer of the adhesion material. The layer of the polycationmaterial provides an exposed face for retaining bioparticles. The methodadditionally comprises performing dielectrophoresis such thatbioparticles contact the exposed face of the layered composition on thetarget surface, whereby the bioparticles are retained on the exposedface of the layered composition at the target surface.

As will be realized, the subject matter described herein is capable ofother and different embodiments and its several details are capable ofmodifications in various respects, all without departing from theclaimed subject matter. Accordingly, the drawings and description are tobe regarded as illustrative and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a prior art technique of trapping P19 cells in amicrofluidic channel using DEP after 24 hours.

FIG. 1B illustrates healthy P19 cells seeded on PEMs in cell growthmedia after 24 hours.

FIG. 2A is a schematic cross sectional illustration of a glass slideassembly in accordance with the present subject matter.

FIG. 2B is a schematic cross sectional illustration of a flow device inaccordance with the present subject matter.

FIG. 3A is an image of a layer of PAH-FITC on a collection of PEMs.

FIG. 3B is an image of a layer of FN-ROX on a collection of PEMs.

FIG. 3C is an image of PAH-FITC deposited on FN-ROX, and both depositedon PEMs.

FIG. 4A is a graph showing the change in the number of cells seeded insucrose media after 24 hours and exposed to PEMs, hCAM, and TOPS.

FIG. 4B is an image showing P19 cells 24 hours after seeding on TOPS.

FIG. 4C is an image showing P19 cells 24 hours after seeding on PEMs.

FIG. 4D is an image showing P19 cells 24 hours after seeding on hCAM.

FIG. 5 is a graph of percent viability of P19 cells on the hCAMcomposition of the present subject matter.

FIG. 6A is an image of P19 cells on adhesive TOPS.

FIG. 6B is an image of P19 cells on (PAH/PSS)₂ FN.

FIG. 6C is an image of P19 cells on hCAM.

FIG. 7 illustrates DEP trapping of P19 cells on hCAM.

FIG. 8A is an image of P19 cell differentiation after DEP trapping.

FIG. 8B is an image of P19 cell differentiation after DEP trapping and 8days in culture.

FIG. 9 is a schematic illustration of a microfluidic device and severalregions detailed, in accordance with the present subject matter.

FIG. 10A is an image of a PET membrane before processing.

FIG. 10B is an image of a PET membrane after processing.

FIG. 11A illustrates another microfluidic device in accordance with thepresent subject matter and a schematic illustration of two fluidsflowing in the device.

FIG. 11B illustrates the device of FIG. 11A and two fluids flowing inthe device.

FIG. 11C illustrates the device of FIG. 11A and two fluids flowing inthe device.

FIG. 11D illustrates the device of FIG. 11A and two fluids flowing inthe device.

FIG. 12 is a schematic illustration of another microfluidic device inaccordance with the present subject matter.

FIG. 13A is an image of a collection of trapped cells on a microelectrode surface in a flow channel.

FIG. 13B is an image of the trapped cells on the microelectrode surface24 hours after trapping.

FIG. 14 is a collection of images illustrating a method of formingelectrically conductive regions on a PET membrane in accordance with thepresent subject matter.

FIG. 15 is a schematic top view of a microfluidic device including a PETmembrane in accordance with the present subject matter.

FIG. 16 is a schematic cross sectional view of the device in FIG. 15taken across line A-A.

FIG. 17 is a collection of images showing cell behavior on an array ofdifferent surfaces.

DETAILED DESCRIPTION OF THE EMBODIMENTS Hybrid Cell Adhesive Materials

Dielectrophoresis (DEP) for cell manipulation has primarily focused onapproaches for separation/enrichment of cells of interest. Advancementsin cell positioning and immobilization onto substrates for cell culture,either as single cells or cell aggregates, has benefited from theintensified research efforts in DEP (electrokinetic) manipulation.However, there has yet to be a DEP approach that provides the conditionsfor cell manipulation while promoting cell function processes such ascell differentiation.

In accordance with the present subject matter, a system is provided thatcombines DEP with a hybrid cell adhesive material (hCAM) to allow forcell entrapment and cell function, as demonstrated by celldifferentiation into neuron-like cells (NLCs). The hCAM, comprised ofpolyelectrolytes and fibronectin (FN), is adapted to function as aninstantaneous cell adhesive surface after dielectrophoretic (DEP)manipulation, and to support long term cell function (cellproliferation, induction, and differentiation). Pluripotent P19 mouseembryonal carcinoma cells flowing within a microchannel were attractedto the DEP electrode surface and remain adhered onto the hCAM coatingunder a fluid flow field after the DEP forces were removed. Cellsremained viable after DEP manipulation for up to 8 days, during whichtime the P19 cells were induced to differentiate into NLCs. Thisapproach could have further applications in areas such as cell-cellcommunication, three-dimensional cell aggregates to create cellmicroenvironments, and cell co-cultures. Although the present subjectmatter and its various embodiments are primarily described with regardto cells, it will be understood that the subject matter is applicable toa wide array of bioparticles.

The term “bioparticle” as used herein refers to any material shed froman organism and is typically biological in nature. The bioparticles canbe classified according to any of the general biological classes ofmaterials. For example, the bioparticle can be proteinaceous (e.g., aprotein, peptide, or antibody), nucleic acid-containing (e.g., anucleobase, nucleotide, oligonucleotide, or nucleic acid),lipid-containing (e.g., fatty acid-containing), steroidal, one or moresmall biological molecules, other types of biological material, andcombinations thereof. Some more specific examples of bioparticlesinclude cells (e.g., skin-derived or epidermis cells), proteinstructures, hair, pathogenic and non-pathogenic bacterial, viral,fungal, protozoal or other organisms, and plant-derived material (e.g.,pollen). Shed material from the skin is particularly plentiful andincludes particles from the outer skin layer (e.g., stratum corneum) andother skin layers that contain keratin. Though the bioparticles arelargely organic, they may also be inorganic. For example, thebioparticle can be a mineral, such as talc. The bioparticle also neednot be natural in composition, but may be synthetic (e.g., particulatesused in cosmetics or other toiletries). Often, the bioparticles areconstructed of aggregations of molecules or other bioparticles. Suchaggregations include cells, viruses, pollen grains, skin flakes, hair,bacteria, and several other types of aggregations of organic andinorganic molecules.

In accordance with the present subject matter, the effects of differentcell adhesive materials on the attachment and function of P19 cells wereassessed to determine the most appropriate surface on which toinvestigate cell function (specifically differentiation) after DEPtrapping and subsequent removal of the electric field. P19 cells are apluripotent cell line that have the ability to differentiate throughseveral pathways in vitro, specifically neuronal, cardiac muscle andskeletal muscle. The ability of P19 cells to differentiate after DEPmanipulation would demonstrate the successful generation of a celladhesive material that allows long term culture. This is critical forperforming experiments with cells that are arranged by DEP. The presentsubject matter demonstrates that an hCAM prepared from FN and apoly(allylamine hydrochloride) (PAH) layer on top of PEMs allowed forinstantaneous cell anchorage after DEP trapping. Furthermore, long termcell viability (more than a week) and differentiation were alsoattained, thus demonstrating the utility of the hCAM for long term invitro cell experiments.

In accordance with the present subject matter, two sets of separateinvestigations were performed to identify a biocompatible coating thatallows P19 cell adhesion and growth under the low conductivity mediasucrose and under electric fields. Tissue culture polystyrene (TOPS),polystyrene (PS) (spin coated plasma oxidized), cell culture media (CCM)pretreated PS, poly-L-lysine, (PAH/PSS)₂PAH, collagen I (Col I), andfibronectin (FN) were evaluated as adhesion substrates for cellssuspended in sucrose for 15 min, the maximum time typically required forDEP positioning of cells. The assessments described herein were based oncounting the number of adhered cells remaining on the surface andcounting the fraction of rounded cells (quantitative cell morphologyassessment) as well as qualitative evaluation of cell morphology versusthe morphology of the cells on the TOPS substrate 24 hours after cellseeding. Previously, it was found that PEMs were able to capture cellstrapped with DEP forces, but the compatibility of PEMs for long termcell culture was not evaluated. The data indicated that the cellsadhered well to the (PAH/PSS)₂PAH when the cells were seeded in cellculture media (700 cells/mm²). But when the cells were seeded insucrose, fewer than 9 cells/mm² were observed. Cell seeding in sucrosesignificantly decreased the number of adhered cells on collagen, PAH,spin coated PS with plasma treatment and fibronectin. Sucrose did notappear to influence adhesion on poly-lysine and TCPS surfaces. Becauseit was found that sucrose did not appear to decrease cell function whenused in tissue culture polystyrene, it was hypothesized that sucrose maydecrease the adhesive nature of the substrate by blocking or removingthe proadhesive molecules. During the substrate evaluation, it was notedthat although cells remain adhered to collagen I and poly-lysine after24 h, greater than 40 and 80 percent of the cells were rounded andappeared unhealthy. Qualitative evaluation of the morphology suggestedthat the P19 cells that remained adhered to the FN substrate had aspread appearance similar to the cells on the TCPS dish. Overall, thedata from this evaluation suggested that the FN substrate best supportsgrowth of the P19 cells when they are seeded from a sucrose solution.

Additional assessments showed that when trapping P19 cells under acontinuous flow field using DEP and FN-coated substrates, all the cellsdetached at the moment the DEP forces were stopped. To take advantage ofthe ability of FN to promote long term adhesion and growth of the P19cells and the PEMs that support capture of cells after DEP forces areapplied, an hCAM composition was prepared from FN, PEMs, and a PAH layeron top of the FN. A schematic of the hybrid material and utilized in twolayered systems is shown in FIGS. 2A and 2B. The combination of FNadsorbed to PEMs (negatively charged PSS as the outermost layer) and PAHon top of the FN was tested for cell adhesiveness under DEP conditionsand long term cell viability. The cell adhesiveness of the hCAM wasassessed first under cell seeding conditions in sucrose. Silanizedcoverslips were polystyrene coated and then plasma oxidized beforedepositing the layers of the polyelectrolytes (see FIG. 2A). Theprocedure to deposit the hCAM was similar for the experiments carriedout on the DEP electrodes (see FIG. 2B), differing only in the PEMsbeing directly deposited on the ITO/glass surface and not on apolystyrene layer. Specifically, a schematic side view of a glasscoverslip assembly 10 is shown in FIG. 2A and a DEP device 110 is shownin FIG. 2B, both with the hCAM deposited on top. The assembly 10 in FIG.2A comprises a glass substrate 12, a layer of polystyrene (PS) 14disposed on the glass substrate, a collection of PEMs 16 disposed on thelayer of polystyrene, and a layer 20 of the hCAM material disposed onthe collection of PEMs. The PEMs 16 are depicted as including four (4)layers, but it will be understood that a greater number or lesser numberof layers could be utilized. The layer 20 of hCAM material includes anunderlayer 22 of FN and an outerlayer 24 of PAH disposed on theunderlayer 22. The flow device 110 and particularly, a microfluidic flowdevice, generally comprises a glass substrate 112, two or moreelectrically conductive electrodes 113 such as indium tin oxide (ITO)electrodes, a collection of PEMs 116 disposed on the glass substrate 112and the one or more electrodes 113, and a layer 120 of the hCAMmaterial. The hCAM material typically includes an underlayer 122 of FNand an outerlayer 124 of PAH disposed on the underlayer 122. The device110 also includes a flow channel 130 which is generally defined by oneor more walls 140 formed from a suitable material such as polydimethylsiloxane (PDMS). The hCAM is comprised of a layer of FN and PAH on topof four layers of polyelectrolytes (PAH/PSS)₂, which in turn weredeposited onto polystyrene-coated coverslips as in FIG. 2A and ITOelectrodes as in FIG. 2B. The microchannel is molded in PDMS andreversibly bound onto the device.

The hCAM was examined by depositing fluorescently labeled FN and PAH inmicrofluidic channels and imaging the channels using fluorescencemicroscopy as shown in FIGS. 3A-3C. Specifically, images offluorescently labeled components of the hCAM are shown in FIGS. 3A-3C.FIG. 3A shows PAH-FITC and FIG. 3B shows FN-ROX deposited on four layersof polyelectrolytes ((PAH/PSS)₂). FIG. 3C shows PAH-FITC deposited onFN-ROX, and both on (PAH/PSS)₂. The interior region in FIG. 3C denotesthe overlapping of the labeled PAH and FN throughout the surface. Thescale bar in FIG. 3C is 200 μm. Fluorescently labeled FN (FN-ROX) andPAH (PAH-FITC) were deposited separately (FIG. 3A and FIG. 3B) and thentogether with PAH-FITC on top of FN-ROX, and in all cases they weredeposited on top of 4 layers of polyelectrolytes ((PAH/PSS)₂). All theimages in FIGS. 3A-3C were taken after the channels were rinsed and thenrefilled with PBS. The fluorescence from PAH-FITC on (PAH/PSS)₂ is shownin FIG. 3A. In FIG. 3A, it was determined that PAH-FITC homogenouslycoats the surface. FIG. 3B shows FN-ROX bound to (PAH/PSS)₂. In FIG. 3B,it was determined that the FN-ROX, also covers the surface of thechannel. FIG. 3C shows PAH-FITC deposited onto FN-ROX, which was firstdeposited on (PAH/PSS)₂. In FIG. 3C, the darker regions (excluding theedges) indicate the areas where there is a thin layer of the materials,whereas the lighter areas are observed at the edges of the channel wheremore accumulation of the deposited PEMs was previously observed. The PBSrinse was performed by aspirating from the outlet reservoir using avacuum pump. The fluorescence intensities remained constant afterrinsing, suggesting that the hybrid layer is stable in a fluid flowfield.

Additionally, the proliferation and viability of P19 cells seeded insucrose on the hCAM surface were assessed. FIG. 4A shows the change inthe number of cells seeded in sucrose media after 24 h. The cells wereexposed to sucrose media for 0 min, 15 min, 30 min, 45 min, and 60 min,after which cell culture media (CCM) was added to the well to substitutethe sucrose media. This plot shows a tendency of the hCAM to allow forsimilar levels of cell proliferation, specifically, cell doubling (seethe crosshatched bars in FIG. 4A) at all time points. The doubling valuewas calculated by dividing the number of cells at 24 hours by the numberof cells seeded at 0 h. A value of 2 is expected if the number of cellsdoubled. Cells exposed to CCM only (0 minutes in sucrose) and sucrosefor 15 min, 30 min, and 45 minutes showed the best results for the hCAMsurface. Only the 60 minutes sample, on the hCAM showed a value of lessthan 2. On the other hand, the cells seeded on PEMs do not exhibit celldoubling except for those that were seeded in CCM (0 minutes insucrose). The average doubling value obtained for P19 cells seeded insucrose on the hCAM and on PEMs were 2.06±0.41 and 1.38±0.25,respectively. These results demonstrate the compatibility of the hCAMwith DEP conditions (sucrose media), which is critical to successfullygenerate DEP trapping forces that will hold cells in place. The PEMalone, on the other hand, was incapable of promoting P19 cell attachmentand proliferation (cell doubling) when cells were suspended in sucrose.

P19 cell morphology after 24 hours was also evaluated, in which FIGS.4B-4D show P19 cells 24 hours after seeding on TOPS, PEMs, and hCAM,respectively. These images show P19 cells that were not exposed tosucrose (FIG. 4B) and cells that were exposed to sucrose (FIGS. 4C and4D) for 15 minutes and later replaced with CCM. The morphology of theP19 cells was affected by sucrose exposure and the surface on which theywere plated. Cells on PEMs appeared more rounded, indicating weakattachment to the surface of the PEMs (see white arrow heads in FIG.4C). In some cases they formed elongated structures larger than theaverage surface area of the cells (see black arrowhead in FIG. 4C).Conversely, P19 cells on hCAM showed similar morphology to the cellsseeded in CCM on TOPS and similar doubling values (doubling value=2.5 onTOPS versus 2.6 on hCAM). Specifically, FIGS. 4A-4D are directed toproliferation of P19 cells seeded on PEMs and hCAM after resuspension insucrose. FIG. 4A shows the change in the number of cells seeded insucrose media after 24 hours (doubling value). Values are approximately2 for cells on the hCAM surface, whereas cells on PEMs show values ofless than 2 when cells were suspended in sucrose (averages of 89cells/frame, 44 cells/frame, and 25 cells/frame were observed for TOPS,PEMs, and hCAM, respectively; error bars represent one standarddeviation). FIG. 4B shows representative phase contrast images of P19cells on TOPS, PEMs (FIG. 4C), and on hCAM (FIG. 4D) 24 hours afterseeding. Cells on PEMs and hCAM were suspended in sucrose for 15 min.Black arrowhead in (FIG. 4C) indicates cell with larger than averagesurface area, and white arrowheads indicate weak cell attachment to thesurface of the PEMs. The scale bar in FIG. 4C is 100 μm.

The viability of P19 cells was assessed using a live/dead viabilityassay from Invitrogen Corp. The viability results in FIG. 5 show that99% or more of the cells were viable 48 hours after cell seeding onhCAM, and 96% of the cells were viable on the PEMs. Also, the resultsindicate that P19 cells can be exposed to sucrose for at least 60minutes with no significant change in viability. Specifically, FIG. 5illustrates percent viability of P19 cells on the hCAM. Cells are 99%viable or more at all sucrose exposure times. The percentage of livecells is represented by the gray color bars, whereas the dead cells(cross hatched bars) complete the 100% of the cells in each bar with ≦1%dead cells.

To fully evaluate the function of P19 cells after 15 minutes of sucroseexposure, adhered P19 cells on TCPS (control, no sucrose exposure),(PAH/PSS)₂/FN, and hCAM were differentiated. Cell differentiation wasevaluated using a procedure that was modified from previous reports onP19 cell differentiation. The present process allows for the plating ofdissociated cells on adhesive surfaces and induction of differentiationafter cell attachment on the substrates. Cell differentiation wascarried out by first exposing P19 cells to sucrose for 15 min,exchanging the sucrose for low serum/retinoic acid induction media andafter 4 days exchanging the low serum media for normal cell growthmedia. FIG. 6A shows an image of immunostained P19 cells that wereinduced to differentiate on adhesive TCPS without exposure to sucrose.Neurofilaments and neurofilament proteins in the cytoplasm of the NLCsare stained with a neurofilament antibody. Neurofilaments are observedas cables connecting the cells, and the arrowheads point atneurofilaments generated by the P19 cells, which differentiated intoNLCs. FIGS. 6B and 6C show P19 cells differentiated on (PAH/PSS)₂FN andon the hCAM, respectively. Each image shows the clear formation ofneurofilaments after P19 cells were induced and differentiated on thesurfaces. This indicates P19 cells can be induced to become NLCs andform neurofilaments even when the cells are fully adhered onto thesesubstrates during the programming and induction process. Specifically,FIGS. 6A-6C show immunofluorescence images of differentiated P19 cellsinduced on TOPS (FIG. 6A), on (PAH/PSS)₂FN (FIG. 6B), and the hCAM (FIG.6C). Neurofilaments were immunostained, demonstrating neuronaldifferentiation (see arrows). Induction and differentiation werepossible while cells were adhered on all surfaces. The scale bar in FIG.6B is 50 μm.

As previously described, previous attempts to use PEMs for long termcell experiments demonstrated that polyelectrolytes alone did notmaintain cell viability after exposure to DEP conditions (sucrose andelectric fields). The hybrid surface of the present subject matter,hCAM, showed the ability to accommodate long term P19 cell growth andfunction after the surface and the cells were exposed to sucrose. Onceit was determined that the hCAM could support long term cell function,the engineered material was used with a DEP device and all conditionsused for such experiments. The combination of polyelectrolytes and FN onthe ITO electrodes produced a surface suitable for DEP-based cellanchorage, proliferation, and differentiation as shown in FIGS. 7 and 8.FIG. 7 shows a sequence of cell movement in a fluid flow field and theapplication of DEP forces.

Specifically, FIG. 7 illustrates DEP trapping of P19 cells on hCAM.Panel A shows P19 cells flow down the channel passing over the DEP ITOelectrodes (vertical dark gray lines) without being trapped. The ITOelectrodes were initially off for a few seconds before they were turnedon. As shown in panel B, once the electrodes were turned on, P19 cellswere trapped by the DEP forces and then anchored onto the hCAM. Panel Cshows ITO electrodes were turned off, and P19 cells trapped on thesurface remained adhered to the hCAM even while exposed continuously toa fluid flow field. The scale bar in Panel 7B is 50 μm. Morespecifically, Panel A in FIG. 7 shows a phase contrast image where P19cells are flowing down the channel (left to right) in the absence ofDEP. The cells are passing over the electrodes without being trapped.The first cells trapped when the DEP forces are active are shown inPanel B of FIG. 7. The applied voltage was varied throughout theinvestigation from 7 V to 3 V at a frequency of 30 MHz (electric fieldsbetween 7,000 V/cm to 3,000 V/cm) in order to start trapping cells onthe first pair of electrodes and later cell trappings on subsequentelectrodes as the voltage was lowered. Panel C in FIG. 7 shows the P19cells trapped at the end of the DEP experiment when voltage is no longerbeing applied. Cells remained trapped under the fluid flow field withoutan electric field present.

Cells that were trapped under DEP conditions on the hCAM surface in afluid flow field were later induced to differentiate into NLCs. FIGS. 8Aand 8B illustrate P19 cell differentiation after DEP trapping and 8 daysin culture in a microfluidic system. The phase contrast image (FIG. 8A)shows a number of neurofilament projections connecting the cells oncethey have differentiated (see arrowheads), which is indicative ofsuccessful P19 cell differentiation into NLCs. The ITO electrodes can beseen (gray vertical lines) in the images where cells grew out afterinitial trapping. Cell migration away from the electrode occurred duringthe 6 days (2 days in CCM and 4 days in induction media) required fordifferentiation.

Immunostaining of neurofilaments better illustrates the complexity ofthe interconnections among the cells. The fluorescence image in FIG. 8Bmore prominently shows the projections P19 cells produced after thedifferentiation process. The presence of stained neurofilament processesand staining in the cytoplasm of NLCs confirms the suitability of thehCAM as a surface that provides for the anchorage of P19 cells under DEPconditions in microfluidics (sucrose media, electric fields, and fluidflow field), and that simultaneously allows the cells to functionproperly in their ability to differentiate after the complete process.

Specifically, FIGS. 8A and 8B depict differentiated P19 cells within amicrochannel after DEP trapping and induction. FIG. 8A shows phasecontrast image of NLCs (differentiated P19 cells) on the hCAM after 8days in the microfluidic system. The vertical dark gray lines are theITO electrodes used to trap the cells on day 1. Arrowheads point to theprojections of differentiated P19 cells. FIG. 8B shows immunostaining ofneurofilaments, a marker of neuronal cells and therefore indicative ofsuccessful differentiation of the P19 cells, illustrates the projectionsfrom differentiated cells throughout the surface of the device. Cells onthe ITO electrodes as well as cells that proliferate away from theelectrode regions differentiated equally. Arrowheads point to theneurofilaments formed during cell differentiation. The scale bar in FIG.8A is 50 μm.

In the subject matter described herein, an engineered cell adhesivesurface was demonstrated with a two-fold purpose: the anchorage of cellsunder DEP conditions with continuous fluid flow field and its ability tosupport long term cell experiments such as cell induction anddifferentiation. A hybrid material comprising polyelectrolytes and FN,with FN and PAH at the surface satisfied this goal. The P19 cells weretrapped with DEP forces and anchored on the hCAM surface in a continuousflow field within a microfluidic system. The cells were viable for up to8 days and were able to undergo neuronal differentiation until cellfixation was carried out for immunostaining purposes. Additionally, theability to induce P19 cells while the cells are adhered to a surface wasdemonstrated. This suggests that neurodevelopment studies that assesscell-cell interactions could be performed in microfluidic devices withhCAM surfaces. Going forward, microfluidics may allow the study ofcell-by-cell mechanisms, including the pattern of morphogen responsetracked by assessing the fraction of cells that have differentiated.This type of study may be possible by integrating DEP investigationsystems with controlled microfluidic laminar flows.

Thus, in accordance with the present subject matter, a hybrid celladhesive material, i.e. hCAM, comprises an outermost layer of one ormore polycation or polyelectrolyte materials disposed on a layer offibronectin (FN) or other extra cellular matrix material. The hCAM layercaptures cells or other bioparticles by attraction fromdielectrophoretic forces, and retains the cells in place along anexposed surface of the hCAM layer. The hCAM layer uses the polycationmaterial(s) to electrostaticallly bind the cells instantaneously whichhave a net negative charge along their surface, while concurrently thefibronectin or other extracellular matrix material promotes long termsurvival of the retained cells.

A wide array of polycation materials can be used in addition to, orinstead of, poly(allylamine hydrochloride) (PAH) such as but not limitedto poly(ethylene imine) (PEI), poly(diallyl-dimethyl ammonium) chloride(PDADMAC), poly(lysine), polyacrylamide (PAAm), similar agents, andcombinations thereof.

A wide array of extracellular matrix materials can be used in additionto, or instead of, fibronectin such as laminin; elastin; collagen;collagen fibrils; proteoglycons such as heparan sulfate, chondroitinsulfate and the like; hyaluronic acid; and any other natural orsynthetic material that will promote cell adhesion and can be assembledin layer-by-layer techniques referred to herein as an extracellularmatrix material anologue. Combinations can also be used.

Preferably, the layers of the hCAM material, i.e. layer(s) of polycationmaterial(s) and layer(s) of adhesion material(s) (i.e. extracellularmatrix), are assembled in a layer-by-layer technique. The term“layer-by-layer” as used herein refers to a strategy by which layers ofmaterials, usually polymers, are stacked one on top of each other byadsorption, and beginning on a substrate. Typically, electrostaticforces keep the layers adsorbed to the substrate initially and then toone another. However, other interactions have been shown to maintain orat least assist in maintaining the multilayers assembled as a singlefilm or layer. Examples of these other interactions include covalentbonds, hydrogen bonding, donor-acceptor interactions, and the like.Generally, if the materials of the hCAM are merely mixed or otherwisecombined, an undesirable precipitate typically forms.

The hCAM can utilize particular concentrations of agents in each of thelayers. When using fibronectin as the extracellular matrix (adhesion)material, a preferred concentration is from about 25 to about 50 μg/ml,based upon the total amount of the FN-containing layer. When using PAHor other similar polycation material, a preferred concentration of thePAH or like material is typically about 1 mg/ml, based upon the totalamount of the polycation layer.

The hCAM can optionally include one or more other agents, components,and/or materials for example polyions and lipids. Moreover, the presentsubject matter contemplates the potential use of particular combinationssuch as fibronectin/heparan sulfate, fibronectin/chondrotin sulfate,laminin/heparan sulfate, and laminin/chondroitin sulfate.

Additional nonlimiting examples of agents that could be included in thehCAM are growth factor peptides and proteins, small molecule drugs, i.e.which are slowly released during cell adhesion and growth, nanomaterials, antibodies (which influence cell response, fluorescentprobes, i.e. for monitoring degradation of the extracellular matrixmaterial, and combinations thereof. Typical concentrations of theseagents in the hCAM are from about 5 μg/ml to about 100 μg/ml.

The total thickness of the hCAM after deposition depends upon the numberof layers, and the distance of the cells to be trapped or retained onthe hCAM layer from the substrate. Typically, at least one or moreunderlying layers are provided on the substrate and are disposed betweenthe substrate and the hCAM. Typically, only one layer of theextracellular matrix material, e.g. fibronectin is needed. That layershould be close enough to the outer exposed surface of the hCAM tosupport cell survival and growth. Although not wishing to be bound toany particular theory, the thickness of the extracellular matrixmaterial, e.g. fibronectin, is generally from about 2 nm to about 5 nm.The thickness of the polycation layer, after drying is typically about 2nm for each layer. Thus, for an assembly of four (4) layers ofpolyions/fibronectin/PAH, the total thickness is generally about 12 nmto about 15 nm.

Polyester Membranes

In accordance with the present subject matter, a new system fordielectrophoretic cell capture on permeable polyester membranes isprovided. Photolithographic techniques were used to fabricate goldmicroelectrodes on a polyester membrane. The characterization of themicroelectrodes showed that there were no differences regardingroughness, permeability, and hydrophilicity of the membrane before andafter processing. Finally, dielectrophoretic cell capture and viabilityin a microfluidic device was demonstrated on the patterned membrane.These membranes could ultimately be combined with multilayermicrofluidic devices to form a powerful tool for studies of cell-cellinteractions in co-culture, whereby spatial separation of different celltypes and/or microenvironments are required.

A multilayer microfluidic device with a PET membrane has been used toseparate the channels for cell culture and cell manipulation to monitorthe induced gene expression of ZsGreen1-DR. The use of permeable PETmembranes in multilayer microfluidic devices has several advantages.Soluble factors could diffuse through the intermediate membrane, andtheir effect on the cells could be observed without disturbinginfluences caused by their supply. Additionally, the double layereddesign adds another level of temporal and spatial control. Thecombination of a multilayer microsystem with dielectrophoretic cellcapturing onto a permeable membrane will enable in vitro co-culturesystems closer to cell-cell interactions that occur in vivo.

In accordance with the present subject matter, the fabrication,characterization, and use of a DEP microfluidic device comprised ofelectrically conductive, e.g. gold, microelectrodes on a permeable PETmembrane is provided. Photolithographic procedures along with othertechniques are used to evaporate and lift-off gold on PET membranes toobtain patterns of interdigitated electrodes. These electrodes werecharacterized using atomic force microscopy (AFM), scanning electronmicroscopy (SEM), and optical microscopy. The electrodes were tested fordielectrophoretic cell capture, and cell viability on the gold and PETmembrane surfaces.

Specifically, the present subject matter provides the fabrication ofgold microelectrodes on a permeable PET membrane. The resulting DEPmicroelectrodes were characterized by several techniques. In FIG. 9, amicrofluidic system 200 in accordance with the present subject matter isshown. The system 200 comprises a plurality of gold electrodes 210. Theinterdigitated microelectrodes 210 shown in the center are linked tocontact pads 220 and 222. FIG. 9, and specifically area B, shows anactual gold/PET surface. The continuous connection of the patterned goldis visible. The system 200 also comprises a PET membrane 230 upon whichare disposed the electrodes 210 and pads 220, 222. The pores of the PETmembrane 230 appear as black spots in the coated as well as uncoatedareas of the surface. SEM images show the surface (area C in FIG. 9),and the insert (area D in FIG. 9). The extent of gold deposition, i.e.with regard to coverage or blockage of the micropores of the PETmembrane, is best illustrated in area D of FIG. 9. SEM imaging showeddark grey spots inside the pores, i.e., the gold did not completelyblock them. The average distance to which the gold was deposited insidethe pores was 2.1 μm±1.2 μm. These observations suggest that the poresremained open and therefore permeable. Even if the partial blockagewould slightly affect the function of the membrane where gold wasdeposited, half of the cell adhesion surface area is not covered by it.Therefore, the permeability of the membrane remained effectivelyunaffected.

Specifically, FIG. 9 illustrates a scheme of the gold pattern and imagesof the gold patterned membrane. Area A shows a typical layout orconfiguration of the microfluidic system 200 having the microelectrodesin the center, which are connected to contact pads. Area B is amicrograph of the processed microelectrodes. Area C is an SEM image ofthe area used to measure the distance to which gold was deposited intothe pores. The pores could be observed throughout the entire membrane.Area D is a detailed view of one pore showing its partial blockage bythe deposited gold. The scale bar in area B is 100 μm, the scale bar inarea C is 3 μm, and the scale bar in area D is 300 nm.

FIG. 10A and FIG. 10B are AFM micrographs of a PET membrane before (FIG.10A) and after processing (FIG. 10B) in accordance with the presentsubject matter. The pores could be observed throughout the membraneregardless of the patterned gold. The coated areas within the patternwere continuously connected to result in interdigitated microelectrodes.In addition, the surface roughness of the PET membrane was measuredbefore and after treatment to assess any possible changes duringprocessing. An RMS (root-mean-square) roughness of 28.7 nm±4.8 nm wasobserved for the membrane before processing, and an RMS roughness of24.3 nm±10.6 nm was observed after treatment. When these results wereanalyzed they showed no statistical difference (ANOVA, analysis ofvariance, single factor, p=0.33). Even the RMS roughness on the goldpattern (27.1 nm±9.6 nm) was not statistically different from the beforeand after processing values mentioned above (p=0.70 and p=0.61,respectively). The mechanical stability of the membrane was visuallyevaluated after processing, whereby no changes were detected. Bothfigures show a surface area of 75 μm×75 μm. The pores are randomlydistributed and have an average diameter of 1.2 μm. The pores can beeasily observed throughout the PET membrane including where thecontinuous layer of gold had been deposited.

As described in Example 3 herein, contact angle measurements were usedto monitor the hydrophilicity of the membrane during the processingsteps (see Table 1 later herein). With a water contact angle of 86° themembrane was slightly hydrophilic before any treatment. The sequentialmicrofabrication steps decreased the contact angle to 69°, whereby thebiggest change occurred after fixing the PET membrane onto the glasswafer via PMMA.

A multilayer microfluidic device 300 (see FIGS. 15 and 16) was assembledto test the permeability of the PET membrane after processing. Themultilayer microfluidic device 300 comprises a glass substrate 310. Abody 320 formed from a suitable polymeric material such as polydimethylsiloxane (PDMS) is disposed on the substrate 310. The body 320 definesat least two flow channels such as a bottom channel 330 and a topchannel 350. The device 300 additionally comprises a PET membrane 340disposed between and generally separating the flow channels 330 and 350.Two different food dyes were exchanged between the two layers bytransporting them through the pores of the membrane (see FIGS. 11A-11D).This ultimately confirmed that its permeability was restored. Details asto this investigation are provided in the description of Example 2herein.

Specifically, FIGS. 11A-11D illustrate permeability testing of the PETmembrane after processing. FIGS. 11A-11D show actual images of light anddark food dyes exchanged between the channels 330, 350 in the multilayermicrofluidic device 300 by their transport through the pores of the PETmembrane 340. Two flow channels 330, 350 were provided for the dyes. Thelight dye flowed from left to right and the dark dye from top to bottom.The lower regions of FIGS. 11A-11D show the corresponding flow patternsof the food dyes in the channels 330, 350. In FIG. 11A, at t=0 both flowrates were 0.5 μl/min, resulting in a mixture at the intersection atmembrane 340. In FIG. 11B, after changing the flow rates (light: 10μl/min, dark: 0 μl/min) the light dye was transported to the top channel350 through the membrane 340 and filled the top channel 350(approximately t=3 min). In FIG. 11C, after inverting the flow rates thedark dye was transported to the bottom channel 330 and filled thechannel (approximately t=7 min). And in FIG. 11D, a combined mixtureoccurred after setting both flow rates back to 0.5 μl/min (approximatelyt=11 min). The scale bars in FIGS. 11A-11D are 100 μm.

The microelectrodes were evaluated for dielectrophoretic cell capture.In order to do this, a microfluidic device was assembled by placing apoly(dimethyl siloxane) (PDMS) microfluidic channel perpendicular to themicroelectrodes. Specifically, referring to FIG. 12, a schematicillustration of the assembled microfluidic device is shown. A piece ofPET membrane with deposited gold electrodes was fixed onto a glasswafer. The location of the microelectrodes is indicated by the square atthe 430. The PDMS microfluidic channel was assembled on top,perpendicular to the microelectrodes. Wires were glued to the contactpads and connected to a waveform generator. Specifically, themicrofluidic device 400 comprises a glass substrate 410, an assembly 420of a PET membrane with electrically conductive electrodes, and at leastone microfluidic channel 450 disposed on the assembly 420 of themembrane and electrodes. The assembly 420 includes contacts such as 422and 424, at which a voltage source is connected such as through wires orother electrical conductors 440, 442.

For the cell trapping experiment NIH-3T3 cells were harvested inlow-conductive media (to perform positive DEP) and inserted into themicrochannel, prefilled with the same media. To avoid cell damage thedielectrophoretic cell capture was carried out within 5 minutes afterharvesting the cells. Trapped cells were collected in about half of themicroelectrode surfaces by varying the applied voltage from 2 V_(p-p) to5 V_(p-p) at a frequency of 10 MHz. Variations in the voltage allowedfor cell capture across the length of the microelectrode array. Whencells experienced higher electric fields they were trapped on the firstfew electrodes. On the other hand, when lower electric fields wereapplied cells tended to be trapped further down on the microelectrodearray. The trapping efficiency could be increased further by eitherusing a highly concentrated cell suspension or longer periods of DEPtrapping. Additionally, the cell trapping efficiency can be influencedby the design of the microelectrodes. By modifying the configuration ofthe electrodes this could be further improved. Most of the trapped cells(approximately 90%) still remained on the PEMs after switching off theDEP forces and exchanging the low-conductive media with cell culturemedia. Cells attached well, as observed in FIG. 13A. A live/dead assaywas carried out 24 hours after cell attachment. The assay showed thatabout 99% of the cells emitted green fluorescence; i.e., these cellswere alive, see FIG. 13B.

Specifically, FIGS. 13A and 13B show efficient cell capture using DEP.FIG. 13A is a micrograph after switching off DEP forces and exchanginglow-conductive media with cell culture media (0 h). NIH-3T3 cell capturewas evident, as soon as 5 minutes from the time the microelectrodes wereenergized. Cells flowed from bottom to top of the figure during DEPtrapping. Arrowheads point at some of the trapped cells. FIG. 13B showsa live/dead staining 24 hours after cell capture was carried out. Thecells spread onto the membrane and green fluorescence could be observedin approximately 99% of the cells, demonstrating that cells were viable.The arrowheads in FIG. 13B point to some of the viable cells. Insertsshow some of the trapped cells in more detail. The scale bars in FIGS.13A and 13B are 100 μm.

FIG. 14 illustrates a method 500 of forming a plurality of goldelectrodes on a PET membrane in accordance with the present subjectmatter. In stage A, a layer 520 of an adhesive such as PMMA is depositedonto a suitable substrate such as a glass wafer 510. Deposition of theadhesive can be performed by a wide array of techniques, such as spincoating. A polyester, e.g. PET, membrane 530 is applied onto a face ofthe adhesive layer 520. In stage B, two layers 540 and 545 ofphotoresist material(s) are applied such as by spin coating onto the PETmembrane 530. In stage C, the photoresist face of the intermediateassembly from stage B is exposed to UV light to form stepped undercuts550 between the two layers 540, 545 of the photoresist materials. Instage D, thin layered regions 560, 565 of gold are deposited on theexposed upwardly directed surfaces of the intermediate assembly fromstage C. Specifically, a collection of lower gold regions 560 and acollection of upper gold regions 565 form. It will be noted that acontinuous layer of gold does not form between the lower and upper goldregions 560, 565 due to the stepped undercuts 550. In stage E, thephotoresist bilayer, i.e. layers 540 and 545, and the upper gold regions565 are removed to thereby leave the lower gold regions 560 on the PETmembrane 530. Additional details related to the method 500, materials,and intermediate and resulting structure are provided in the descriptionof Example 2 herein.

FIG. 17 is a collection of images illustrating cell behavior at variousstages and on different surfaces. The details of this investigation areprovided in the description of Example 3 herein.

DEP has been widely used in microfluidic platforms. The choice ofplatform will depend on the evaluations to be carried out. The type ofbioparticle (e.g., cells and viruses) to be manipulated definesguidelines such as the design of the electrodes or performingexperiments with or without flow. For example, hematopoietic tumor cellswere analyzed using a DEP system without applied flow. The electrodesgenerated cell trapping forces and at the same time createdelectro-thermal vortices that produced efficient drug mixing, allowingfor the analysis of cancer drug-induced cytotoxicity. A similarexperiment was carried out where hepatitis A viruses were trapped in amicrosystem using electro-hydrodynamic flow and DEP forces. These kindsof systems use non-adherent bioparticles and therefore provide platformsthat can usually be reused several times. However, studies usingadherent cells mostly require cell adhesive molecules on top of theelectrodes to allow cell behavior and, hence, cell responses that wouldprovide meaningful data. Since cells tend to rearrange the adhesivemolecules they attach onto and leave behind residues from their ownextracellular matrix after detaching, the number of times the devicescan be reused is limited. When cells are used in a microfluidic platformit is beneficial to have some form of trapping mechanism. However, theuse of, for example, mechanical traps creates areas with different flowvelocities, hence influencing the flow near the cells. This could likelyaffect the results of experiments in the cases where cells are sensitiveto such shear forces. In contrast, DEP systems with planar electrodesrender a channel without features that disturb the flow.

The results demonstrate the functionality of the patternedmicroelectrodes on the permeable PET membrane for dielectrophoretic cellcapture. This membrane along with DEP would be suited for specializedapplications such as studies of drug transport, cell monolayerpermeability and cell co-cultures, among others. However, theseapplications would gain the most when combined with multilayermicrofluidic devices. The added levels of control and the benefit of thelocalized cell enrichment by DEP trapping are at the heart of thesedevices. In addition, the combination of DEP and PEMs on a permeable PETmembrane allows fast and reliable cell capture at a high efficiency, andhence subsequent long term cell culture is achievable.

The present subject matter provides the use of gold or otherelectrically conductive microelectrodes on PET membranes as substratesto perform DEP cell entrapment in a microfluidic device. Themicroelectrodes for DEP were fabricated using conventionalphotolithographic and metallization processes. The membrane wascharacterized with different techniques, and results showed that therewas no difference in terms of hydrophilicity, roughness, andpermeability of the membrane when comparing the before and afterprocessing surfaces. Finally, it was demonstrated that the patternedelectrodes can be used for DEP cell trapping experiments in amicrofluidic channel. The cell viability assessment showed that cellswere viable 24 hours after DEP trapping, demonstrating that long termcell experiments can be carried out. This approach allows for an easyand rapid way of cell entrapment and enrichment onto PET membranesurfaces. By combining this work with multilayer microfluidic devices anew platform for cell-cell interactions or cell co-culture studies couldbe developed. Cell exposure to different microenvironments would bepossible, having two cell types physically separated. Future work willfocus on the use of these membranes in multilayered microfluidic systemsfor cell-cell interaction studies.

EXAMPLES Materials Example 1

Poly(allylamine hydrochloride) (PAH, MW=70,000), PAH-Fluoresceinisothiocyanate (FITC), monoclonal anti-neurofilament antibody producedin mouse, antimouse IgG-FITC, retinoic acid 98%, FN, sucrose,poly-L-lysine, and polystyrene pellets were purchased from Sigma-Aldrich(St. Louis, Mo.). Poly(styrenesulfonic acid) (PSS, MW=70,000) waspurchased from Polysciences, Inc. (Warrington, Pa.).Poly(dimethylsiloxane) (PDMS, Sylgard 184) was purchased from DowCorning (Midland, Mich.). Alpha Minimum Essential Medium (αMEM) withribonucleosides and deoxyribonucleosides, calcein AM, ethidiumhomodimer-1, 6-carboxy-X-rhodamine, succinimidyl ester (6-ROX-NHS), andfetal bovine serum (FBS) were obtained from Invitrogen Corporation(Carlsbad, Calif.). P19 cells, 0.25% trypsin-ethylenediaminetetraaceticacid (EDTA) and calf bovine serum (CBS) were purchased from ATCC(Manassas, Va.). Purecol (acidified bovine collagen I) was purchasedfrom Advanced BioMatrix (San Diego, Calif.). Dulbecco'sphosphate-buffered saline (DPBS) and phosphate-buffered saline (PBS)were obtained from Mediatech, Inc. (Hernon, Va.). Indium tin oxide(ITO)/glass substrates were purchased from Delta Technologies(Stillwater, Minn.), and 22 mm×22 mm #1.5 Corning coverslips wereobtained from Daigger (Vernon Hills, Ill.). Electrically conductiveadhesive was purchased from Epoxy Technology Inc. (Billerica, Mass.).Octyldimethylchlorosilane was obtained from Gelest (Morrisville, Pa.).SU-8 photoresist and developer were obtained from MicroChem Corp.(Newton, Mass.).

Cell Culture

P19 cells were cultured in αMEM with ribonucleosides anddeoxyribonucleosides. The growth medium was supplemented by adding 7.5%of bovine calf serum and 2.5% fetal bovine serum (37.5 mL and 12.5 mL ina total of 500 mL of αMEM, respectively). Growth medium was renewedevery 2 d, and cells were subcultured every 2 days to 3 days at adilution ratio of 1:10. Cells were maintained in a humidifiedenvironment with 5% carbon dioxide and a temperature of 37° C.

Coverslips were cleaned with isopropyl alcohol (IPA) using a lint-freecloth wipe and were blown dry with compressed nitrogen (N₂) beforeplacing them flat on a glass Petri dish. An 8 mg/mL polystyrene (PS)solution prepared in toluene was spin coated (418.9 rad/s, 50 s) ontothe coverslips, and the PS-spin coated coverslips (PS thickness between55 nm to 85 nm) were placed in a vacuum chamber for 3 hours to removeany residual solvent. All PS-coated coverslips were plasma oxidizedprior to cell adhesive material deposition.

Cells were seeded in sucrose and in cell culture media (CCM) oncoverslips coated with natural or synthetic materials. Incubation timeswere different for each material and pretreatment. Coverslips pretreatedwith CCM prior to cell seeding were incubated with the CCM for 1 hour at37° C. Additional coverslips were incubated with Collagen I (Col I, 30μg/mL), poly-L-Lysine (1 mg/mL), and FN (25 μg/mL to 50 μg/mL) for 90minutes at 4° C. PAH and PSS solutions (1 mg/mL, mol/L (M)concentrations of the repeating units: PAH=10.7 mmol L⁻¹ and PSS=4.8mmol L⁻¹) were each prepared in 18.2 MΩ filtered deionized water(DI-water). The pH of the PAH and PSS solutions was adjusted to 5 and 6,respectively. Four alternating PEMs, (PAH/PSS)₂, were deposited onto theoxidized PS surface of the coverslips by immersing the coverslip in thepolyelectrolyte solutions sequentially. The initial PAH layer wasdeposited for 40 min. The coverslip surface was rinsed with DI-watertwice before applying subsequent alternating layers for 10 minutes withtwo DI-water rinses between each incubation. After the fourth layer wasdeposited, the PAH outermost layer (fifth layer) was deposited for atleast 30 minutes at room temperature (approximately 21° C.±2° C.). P19cells were then seeded in a 0.32 mol/L sucrose solution for 15 minutesat room temperature, and then the sucrose was aspirated and CCM wasadded to the cells. Images at 0 hours (after adding CCM at the end ofsucrose exposure) and 24 hours were taken to assess the morphologydifferences and adherent status of the seeded cells. The number of cellsadhered to the substrates and the number of rounded (i.e. unhealthy)cells were determined with ImageJ software and the surface that had thehighest number of cells with the lowest number of rounded cells wereselected for use in the DEP device.

Coverslips for the deposition of the hCAM were prepared using the sameprocedure previously described, but to promote better adhesion of thespin-coated polystyrene the following silanization step was added priorto spin coating the polystyrene. Cleaned coverslips in a Petri dish wereplaced in a dessicating chamber containing a Teflon holder with 200 μLof octyldimethylchlorosilane. House vacuum was applied to the chamberfor 2 h, and then the Petri dish was placed in a 60° C. oven for atleast 3 h. All PS-coated coverslips were plasma oxidized prior to PEMdeposition. PEMs were deposited as described in the previous section,except that after the fourth layer was deposited, the wells were rinsedtwice and then stored overnight with DI water at room temperature.

The PEM coated coverslips were then incubated in a 50 μg/mL solution ofFN prepared in DPBS at 4° C. for 1.5 h. The coverslips were rinsed twicewith PBS, and the final hCAM layer was deposited by incubating thecoverslips in 1 mg/mL PAH for 45 minutes at 4° C. The hCAM coverslipswere rinsed twice with DI-water and then transferred to PBS in a newwell in a 6-well cell culture plate until cell seeding.

The hCAM was deposited on the ITO electrode substrates as describedabove, except it was applied in a microfluidic polydimethylsiloxane(PDMS) channel covering the DEP electrodes. In this case, the solutionswere added to the channel inlet and flowed down the channel previouslyaligned onto the DEP electrodes. Once each deposition was completed, thesolutions were aspirated via the channel outlet. The incubation timesand the concentration of the solutions remained the same.

A 0.32 mol/L (M) sucrose solution was prepared in DI-water to mimic theosmolarity of the P19 cell culture media but with low electrolyteconcentration to maximize DEP forces. Confluent (80%) P19 cells weretrypsinized with 0.25% trypsin-EDTA, and were divided into two 15 mLcentrifuge tubes. The cells were centrifuged for 7 minutes at 83.8 rad/sand 5° C. At this point the cells were ready for incubation with sucroseat different time points (0 min, 15 min, 30 min, 45 min, 60 min). Forthe 0 minutes sample, one tube of cells was resuspended in cell culturemedia, and the cells were seeded onto the hCAM coverslips at a dilutionratio of 1:10 (approximately 4700 cells/cm²). The second tube of cellswas resuspended in the sucrose solution, and the cells were seeded ontothe same substrate at an identical cell seeding density (approximately4700 cells/cm²). After each sucrose incubation time point, 4 mL of cellgrowth media was added to the samples to dilute the sucrose (a 1/27dilution, 3.7 final sucrose solution) and restore to normal cell cultureconditions. Phase contrast images of the P19 cell growth on the hCAMwere taken at 0 h, 24 h, and 48 h.

P19 cell viability on the hCAM surface was assessed after 48 hours usingthe LIVE/DEAD viability assay kit from Invitrogen Corp. Calcein AM(excitation/emission maxima at 495 nm/515 nm) was used to stain theviable cells, which exhibit intracellular esterase activity, whileethidium homodimer-1 (EthD-1) (excitation/emission maxima at 495 nm/635nm) was used to label dead cells with damaged plasma membranes.

Calcein AM and EthD-1 were diluted to 2 μmol/L and 4 μmol/L,respectively, in a single solution in DPBS. 1 mL of the dye solution wasadded to each well, and the 6-well plates were placed in the incubatorfor 45 min. The cells were imaged immediately using phase contrastoptics and FITC and Rhodamine filter sets. The images were taken intriplicates for each time point. Viable and dead cells were countedmanually, and the percentage of each was expressed based on the totalnumber of cells per frame. A minimum of 440 cells, per frame, werecounted.

SU-8 masters with raised features (30 μm to 35 μm height, 1000 μm wide)for molding PDMS microchannels were fabricated using the manufacturer'sprotocol. PDMS microfluidic structures were molded by pouring thepolymer on the SU-8 master and curing at 100° C. for 1 hour (frommanufacturer's product information sheet).

ITO electrodes were made from ITO/glass substrates. The ITO surface waspatterned using conventional photolithographic methods. A negativephotoresist was spin coated on the ITO surface and then exposed to UVlight through a chrome mask containing the electrode design. The patternwas developed, and the exposed ITO was etched using a 9 mol/L (M)hydrochloric acid (HCl) solution. The remaining ITO pattern was thenexposed by dissolving the remaining photoresist on the substrate. Wireconnections were made by bonding silver/copper wires to ITO pads usingan electrically conductive adhesive (H37-MPT, Epoxy Technology, Inc.)heated at 150° C. for 1 hour (from manufacturer's product data sheet).

P19 cells were detached from the cell culture surface by trypsinization,centrifuged at 83.8 rad/s for 7 minutes at 5° C., and then resuspendedin 0.32 mol/L sucrose. The cells were immediately introduced into themicrofluidic channel covering the electrodes via the inlet reservoir.Approximately 150 μL of the cells resuspended (approximately 375,000cells) in sucrose were added to the inlet that accessed the channelpreviously filled with the sucrose solution. A flow was produced whenthe cells were introduced due to the difference in pressure between theinlet and outlet reservoirs. Once the cells started to flow down thechannel, a sine wave of up to 7 V_(p-p) was applied at a frequency of 30MHz. The cells were exposed to the DEP forces for up to 4 minutes atwhich point the DEP electrodes were de-energized. Then, thecells/sucrose solution in the inlet reservoir was exchanged for cellculture media to replace the sucrose in the channel. The DEP device withthe trapped cells in cell culture media was then placed in the incubatorset at 37° C. and 5% CO₂.

The cells were maintained by adding fresh cell culture media to theinlet reservoir every 24 h, and by removing the media collected in theoutlet or waste reservoir at the same time. Images of the cells weretaken every 24 h.

P19 cells are typically induced in suspension. However, the presentapproach requires the induction procedure to be carried out on a surface(hCAM) rather than in suspension. Therefore, P19 cells were firstinduced on tissue culture polystyrene (TOPS) to determine if it wasfeasible to induce them on a surface, and then the cells were induced on(PAH/PSS)₂/FN and hCAM. The results on the three surfaces were thencompared. Induction on all surfaces was carried out using the sameconditions in terms of chemicals and days of induction. The onlydifference was the surface onto which the P19 cells were attached. Theprocedure that follows applied to all inductions. To induce thedifferentiation of P19 cells, the CCM was replaced by induction media(IM) comprised of ∞MEM supplemented with 5% FBS and retinoic acid at afinal concentration of 0.5 μmol/L. IM was changed every 24 hours for aperiod of 4 days. On day 4, the IM was replaced with CCM, which in turnwas replaced every 24 hours for two days. Cell differentiation wasverified by using a fluorescently labeled antibody to stain for markerproteins associated with neuronal differentiation two days after cellinduction was completed.

Differentiated P19 cells were fixed by first rinsing the cells with PBSand then adding 4% paraformaldehyde in PBS. The fixation was allowed tooccur at room temperature for 10 minutes at which time the cells wererinsed with PBS. Cells were then permeabilized with a solution of 0.25%Triton X-100 in PBS and then incubated with the primary antibody(monoclonal antineurofilament) at a dilution of 1:40 for 3 hours at roomtemperature. The samples were rinsed with PBS and incubated at roomtemperature with a secondary antibody (antimouse IgG-FITC, Cat. No.F9137, Sigma-Aldrich) at a dilution of 1:200 for up to 90 min. Theneurofilament staining was observed with a 200M Zeiss microscope using amercury lamp source and a filter set with a band pass for excitation at450 nm to 490 nm, a dichroic beam splitter at 510 nm, and a band passfor emission at 515 nm to 565 nm. The objective used had a 10×magnification and an aperture of 0.3. Images were taken using a ZeissMRm camera.

Example 2

Fabrication of gold electrodes on a PET membrane is shown in FIG. 14.The PET membrane was first fixed onto a glass wafer using poly(methylmethacrylate) (PMMA) as adhesive, to prevent folding. Goldmicroelectrodes were fabricated on top of the PET membrane usingconventional photolithographic and metallization techniques. Theresulting microelectrodes were characterized by AFM, SEM, and opticalmicroscopy. Polyelectrolyte multilayers (PEMs) were deposited onto thesurface of the PET membrane containing the microelectrodes in order totrap and anchor cells. Subsequently, the microfluidic device wasassembled and the microelectrodes were tested for cell capture byapplying DEP forces. Cell viability was assessed 24 hours after cellcapture.

Fixation of the PET membrane (11 μm thick, 1.2 μm pore size, 1.6×10⁶pores per cm², cell culture treated, it4ip, Belgium) was necessary forphotolithographic processing to prevent folding of the membrane andhence to avoid problems with the gold patterning. Therefore, 495 PMMA A11 (MicroChem, Newton, Mass.) was spin coated onto a 4 inch (10.16 cm)glass wafer (Valley Design Corp., Shirley, Mass.) to a thickness of 2.25μm. A piece of PET membrane of about 2 cm×2 cm was placed in the middleof the wafer, and then the wafer was baked at 110° C. for 5 min. For thebilayer lift-off process two different photoresists were spin coatedonto the membrane. First, the membrane was coated with the lift-offresist LOR 3A (MicroChem, Newton, Mass.) to a thickness of 350 nm andbaked at 155° C. for 10 min. Second, the positive tone photoresist S1813(Rohm & Haas, Marlborough, Mass.) was spun to a thickness of 1.2 μm andbaked at 110° C. for 5 min. Next, the wafer was exposed to UV-light(A=405 nm) for 5 seconds (MA/BA6, SUSS MicroTec AG, Garching, Germany)to transfer the pattern of the microelectrodes (1000 μm long and 10 μmwide with gaps between opposite electrodes of 10 μm) onto thephotoresist. Finally, the pattern was developed in MF-319 (Rohm & Haas,Marlborough, Mass.) for 60 s. Afterwards, the wafer was placed overnightunder vacuum to allow complete drying of the membrane.

A 50 nm thick layer of gold was deposited onto the photolithographicallyprocessed wafer (E-bream evaporator Denton Infinity 22, Denton VacuumLLC, Moorestown, N.J.). Redundant gold was lifted-off in 1165 remover(MicroChem, Newton, Mass.). To support and accelerate the process,agitation and short pulses of sonication (3 seconds to 5 seconds) wereapplied. The lift-off was completed within 5 minutes to 10 minutes.After the lift-off process the sample was blow dried. The dimensions ofthe microelectrodes after all processing steps varied slightly from thedesign pattern (electrodes widths of approximately 11 μm and gaps ofapproximately 9 μm).

The distance to which gold was deposited into the pores was assessed byimaging a total of 10 pores, randomly selected, with field-emission SEM(Ultra-60 FESEM, Zeiss, Germany). To obtain the distance to which goldwas deposited into the pores, the difference in the working distances oftwo SEM images in the same pore were measured: the first image wasfocused on the surface of the membrane (as outer value), and the secondone was focused on the deepest point inside the pore where gold wasstill seen (as inner value). The difference between the inner and outerworking distances corresponded to the distance to which the gold wasdeposited inside the pore.

AFM images (Dimension 5000, Digital Instruments, Santa Barbara, Calif.)were acquired in tapping mode. Images were acquired at ambientconditions on dry samples. To obtain the differences in RMS roughness ofthe surfaces within the samples, seven independent areas of 10 μm×10 μmwere imaged and then analyzed using the Nanoscope 7.3 software. Theanalyzed surfaces were: 1) membrane before processing; 2) membrane afterprocessing; and 3) the gold patterned.

Contact angles were measured to characterize the hydrophilicity of thePET membrane during the processing. A drop of water was placed onto thesample, and a side view picture was taken. The droplet curvature wasfitted using the software FTA32 (First Ten Angstroms, Inc., Portsmouth,Va.) to obtain the contact angle value. A contact angle between 0° and90° was defined as a hydrophilic surface and a value between 90° and180° as a hydrophobic surface. For each step during the electrodemicrofabrication the contact angle was averaged from four independentmeasurements.

The SU-8 master (SU-8 2025, MicroChem, Newton, Mass.) contained featuresfor molding a microchannel out of PMDS (Sylgard 184, Dow Corning,Midland, Mich.) with a height of 30 μm and a width of 1000 μm. It wasfabricated using the manufacturer's protocol. PDMS was cured on the SU-8master after mixing the elastomer and curing agent at a ratio of 10:1,respectively. Once mixed and degassed, the mixture was poured onto theSU-8 wafer and cured for 4 hours at 65° C. Excessive PDMS was cut, andaccess holes of approximately 5 mm were punched. The PDMS microchannelwas rinsed with 70% ethanol before it was assembled onto the substrate.

A multilayer microfluidic device as shown in FIGS. 15 and 16, wasassembled to confirm that the permeability of the PET membrane was notaffected during processing. The membrane was transferred onto a PDMSlayer containing the top channel. Next, the PMMA was dissolved inacetone. The PDMS layer containing the membrane was plasma activatedalong with the PDMS layer containing the bottom channel. The membranewas sandwiched between both PDMS layers to complete the assembly of thedevice. Both layers contained a microfluidic channel of 30 μm in heightand 1000 μm in width. These channels were perpendicularly aligned toeach other, whereby the intermediate PET membrane allowed the exchangeof reagents. Tubing was connected between the assembled microchip andsyringe pumps (PHD 2000, Harvard Apparatus, PA). For the permeabilitytest the flow rates varied between 0, 0.5 and 10 μl/min. Specifically,referring to FIGS. 15 and 16, the processed PET membrane was alignedbetween the channels of the top and bottom PDMS layers. FIG. 15indicates the flow directions in both channels. The assembled device iscomprised of a glass substrate, the bottom PDMS layer, the membrane, andthe top PDMS layer. The dashed line in the top view denotes the positionat which the cross section of FIG. 16 is taken. The cross sectionindicates the area where the exchange of reagents between the channelswas possible, only through the pores of the PET membrane (see arrows).

To enable cell anchorage and cell culture on chip afterdielectrophoretic cell capture, the area of the membrane containing themicroelectrodes was coated with PEMs as described in Reyes et al.Briefly, 5 μL of a 1 mg/mL poly(ethyleneimine) solution (MolecularWeight (MW)=70000, Polysciences, Inc., Warrington, Pa.) were placed onthe microelectrodes. This first layer was incubated for 30 min, rinsedwith water, and blow dried. Next, two bilayers of polyanion/polycationwere deposited (polyanion=sodium poly(styrene sulfonate), MW=70000,Polysciences, Inc., Warrington, Pa.; and polycation=poly(allylaminehydrochloride), MW=70000, Sigma-Aldrich Corp., St. Louis, Mo.; 1 mg/mLeach). Each layer was incubated for 10 min, rinsed with water, and blowdried. These procedures resulted in the deposition of a total of fivelayers of polyions on the microelectrodes.

NIH-3T3 mouse embryonic fibroblast cells were cultured in DMEM(Dulbecco's Modified Eagle's Medium) modified with 10% (v/v) bovine calfserum. Media was replaced every other day, and cells were subculturedwhen they were 80% confluent using 0.25% (w/v) trypsin (all reagentsfrom ATCC, Manassas, Va.). For DEP experiments the cells were harvestedin 0.147 mol/L sucrose (Sigma-Aldrich Corp., St. Louis, Mo.). Sucrose, anon-electrolyte, was used as low-conductive media to perform positiveDEP, i.e., the cells were attracted by the DEP forces.

Wires were connected to the contact pads using an electricallyconductive adhesive (Epoxy Technology Inc., Billerica, Mass.), cured for1 hour at 150° C. Then, the membrane was coated with PEMs as previouslydescribed herein. Finally, the device was assembled by placing thecleaned PDMS microchannel on top of the microelectrodes, so that thechannel was perpendicular to the microelectrodes. The assembled devicewas connected to a waveform generator (Agilent Technologies, SantaClara, Calif.), and the channel was filled with 0.147 mol/L sucroseusing capillary forces. 150 μL of the cell suspension were placed intothe inlet of the microchannel. Suction was applied from the outlet tostart the cell flow (linear velocity of approximately 550 μm/s). Cellswere captured by applying a sine wave from 2 V_(p-p) to 5 V_(p-p) at afrequency of 10 MHz for less than 5 min. Subsequently, the cell/sucrosesolution in the inlet was exchanged with cell culture media, and thedevice was placed in the incubator at 37° C. and 5% CO₂. After 24 hoursa live/dead assay (Live/Dead® viability/cytotoxicity kit, Invitrogen,Eugene, Oreg.) was performed as described in the manufacture's protocol.Briefly, before imaging, the cells were incubated in media containing 2μmol/L Calcein AM and 4 μmol/L Ethidium homodimer-1 for 20 min. Greenfluorescence indicated living cells and red fluorescence dead ones.

Additionally, cell adhesion and viability of NIH-3T3 cells was testeddirectly on glass, on PEMs on glass and in a cell culture flask.Therefore, the cells were seeded onto these surfaces and incubated for24 hours at 37° C. and 5% CO₂. Afterwards a live/dead assay wasperformed as described above.

Example 3

Water contact angles were measured at different points during thefabrication process to monitor changes in hydrophilicity of the PETmembrane. The results of this evaluation are presented below in Table 1.

TABLE 1 Water contact angles of the PET membrane (n = 4). Point ofMeasurement Contact Angle [°] before processing 86 ± 1 on PMMA 74 ± 1after development 73 ± 1 after lift-off 69 ± 2

To compare the NIH-3T3 cell behavior on our PEMs/PET membrane withstandard cell culture, cell adhesion and viability on other surfaces,i.e., directly on glass, were assessed for PEMs on glass and on cellculture flask polystyrene (FIG. 17). A live/dead assay revealed about99% of living cells after 24 h. The cells showed similar behavior whenseeded on the other surfaces to cells on the PET membrane, i.e., thedifferent surfaces have no influence on cell adhesion and viability.Specifically, FIG. 17 shows cell adhesion and viability on standard cellculture surfaces. The behavior of NIH-3T3 cells did not significantlydiffer on the various surfaces. After 24 hours approximately 99% of thecells were alive. The scale bars in the squares are each 50 μm.

Many other benefits will no doubt become apparent from futureapplication and development of this technology.

All patents, applications, and articles noted herein are herebyincorporated by reference in their entirety.

As described hereinabove, the present subject matter overcomes manyproblems associated with previous strategies, systems and/or devices.However, it will be appreciated that various changes in the details,materials and arrangements of components, which have been hereindescribed and illustrated in order to explain the nature of the presentsubject matter, may be made by those skilled in the art withoutdeparting from the principle and scope of the claimed subject matter, asexpressed in the appended claims.

1. A layered composition for capturing cells and bioparticles duringdielectrophoresis (DEP), the layered composition comprising: at leastone layer of an adhesion material; and a layer of a polycation materialdisposed on at least one layer of the adhesion material, the layer ofthe polycation material providing an exposed face for capturing cellsand bioparticles during dielectrophoresis.
 2. The layered composition ofclaim 1 wherein the polycation material is selected from the groupconsisting of poly(allylamine hydrochloride) (PAH), poly(ethylene imine)(PEI), poly (diallyl-dimethyl ammonium) chloride (PDADMAC),poly(lysine), polyacrylamide (PAAm), and combinations thereof.
 3. Thelayered composition of claim 2 wherein the polycation material ispoly(allylamine hydrochloride) (PAH).
 4. The layered composition ofclaim 1 wherein the adhesion material is selected from the groupconsisting of fibronectin (FN), laminin, elastin, collagen, collagenfibrils, proteoglycans, hyaluronic acid, an extracellular matrixmaterial analogue, and combination thereof.
 5. The layered compositionof claim 1 wherein the layer of the polycation material is adsorbed onthe layer of the adhesion material. 6.-15. (canceled)